Activation of phospholipase D involved in both injury and survival in A549 alveolar epithelial cells exposed to H2O2

To determine the role of the phospholipase D (PLD) pathway in injury and survival of alveolar epithelial cells, A549 cells were exposed to H₂O₂ (500 μM) which resulted in time-dependent injury and bi-phasic increase of PLD activity at 5 min and at 3 h, respectively. n-Butanol (0.5%) inhibited PLD activation, attenuated cell injury at 5 min of H₂O₂ exposure, but enhanced injury at 3 h of exposure. This activation was inhibited by treatment with catalase (500 units/ml). Exogenous phosphatidic acid mimicked the effects of PLD activation, and diphenyliodonium (NADPH oxidase inhibitor) reversed the decline in cell viability induced by H₂O₂ exposure. Propranolol (phosphatidic acid phospholydrolase inhibitor) and quinacrine (phospholipase A2 inhibitor) had weak effects on H₂O₂-induced PLD activation but reversed H₂O₂-induced injury. We speculate that PLD activation at the initiation of H₂O₂ exposure predominantly results in NAPDH oxidase activation, which mediates A549 cell injury, but turns to mediating cell survival as the H₂O₂ attack continues, which might be mainly due to the accumulation of intracellular phosphatidic acid.     

Ochratoxin A induces G2 phase arrest in human gastric epithelium GES-1 cells in vitro

Ochratoxin A (OTA) is a mycotoxin commonly found in several food commodities worldwide. OTA exposure was involved in the nephrotoxicity, hepatotoxicity as well as immunotoxicity in experimental model. Our previous study showed that the high level of OTA contamination in wheat might be related to the high incidence of gastric carcinoma in rural area of China. However, there were no available data regarding the gastric toxicity of OTA up to now. In the present study, we explored the toxicity of OTA in human gastric epithelium immortalized cells (GES-1) by analyzing the regulation of the cell cycle, apoptosis and its molecular mechanism. We found that OTA could induce GES-1 cells arrested in G₂/M phase. Among these cycle-arrested cells, the proportion of cells in M phase was down-regulated after OTA treatment by the mitotic index and the level of phospho-histone H3. Thus, it was clear that OTA exerted a major influence on G₂ phase arrest instead of M phase. We further detected the expression of the key factors which are critical to the G₂/M phase transmission such as Cdc25C, Cdc2 and cyclinB1. The cyclinB1–Cdc2 complex was reduced and the expression of Cdc25C, Cdc2 and cyclinB1 were significantly decreased by OTA treatment both at protein and mRNA level, respectively. Considering that the cells may undergo apoptosis or death due to the cell cycle arrest, so we next detected the apoptosis of cells by OTA treatment. The results confirmed that OTA did induce apoptosis of GES-1 cells and activate the cleavage of capase-3. In conclusion, cell apoptosis and G₂ phase arrest mediated by Cdc25C, Cdc2 and cyclinB1 may be the initiating event in the gastric toxicity of OTA.     

Gene expression changes in primary human nasal epithelial cells exposed to formaldehyde in vitro

Using various exposure conditions, we studied the induction of DNA-protein crosslinks (DPX) by formaldehyde (FA) and their removal in primary human nasal epithelial cells (HNEC). DPX were indirectly measured by the alkaline comet assay as the reduction of gamma ray-induced DNA migration. DPX are the most relevant primary DNA alterations induced by FA and the comet assay is a very sensitive method for the detection of FA-induced DPX. In parallel experiments, we investigated changes in gene expression by using a full-genome human microarray. After a single treatment with FA (50–200 μM), concentration- and time-dependent changes in gene expression were seen under conditions that also induced genotoxicity. Repeated treatments with low FA concentrations (20 and 50 μM) did not lead to a significant induction of DPX but repeated treatments with 50 μM FA changed the expression of more than 100 genes. Interestingly, altered expression of genes involved in the main pathways for FA detoxification and the repair of DPX were not specifically detected.     

M3受体对体外H2O2诱导大鼠心肌细胞凋亡的保护作用

目的探讨M₃受体激动对H₂O₂诱导的大鼠培养心肌细胞凋亡的作用，进一步阐明其机制。方法末端标记法 (TUNEL)进行细胞凋亡检测；免疫组化方法检测Bcl-2和Fas的表达；共聚焦显微镜观察［Ca²⁺］_i荧光强度变化。结果M₃受体激动剂胆碱(10 mmol·L^-1)可减少H₂O₂诱导的心肌细胞凋亡的数量，并可增加心肌Bcl-2的表达，减少Fas表达，抑制H₂O₂诱导的［Ca²⁺］_i荧光强度的升高。但预先应用4DAMP (10 nmol·L^-1)阻断M₃受体可逆转胆碱作用。结论激动M₃受体对H₂O₂诱导的心肌细胞凋亡有保护作用，其机制可能与Bcl-2和Fas表达以及下调［Ca²⁺］_i有关。     

Role of phospholipases A2 in diabetic retinopathy: In vitro and in vivo studies

Diabetic retinopathy is one of the leading causes of blindness and the most common complication of diabetes with no cure available. We investigated the role of phospholipases A₂ (PLA₂) in diabetic retinopathy using an in vitro blood–retinal barrier model (BRB) and an in vivo streptozotocin (STZ)-induced diabetic model. Mono- and co-cultures of endothelial cells (EC) and pericytes (PC), treated with high or fluctuating concentrations of glucose, to mimic the diabetic condition, were used. PLA₂ activity, VEGF and PGE₂ levels and cell proliferation were measured, with or without PLA₂ inhibition. Diabetes was induced in rats by STZ injection and PLA₂ activity along with VEGF, TNFα and ICAM-1 levels were measured in retina. High or fluctuating glucose induced BRB breakdown, and increased PLA₂ activity, PGE₂ and VEGF in EC/PC co-cultures; inhibition of PLA₂ in mono- or co-cultures treated with high or fluctuating glucose dampened PGE₂ and VEGF production down to the levels of controls. High or fluctuating glucose increased EC number and reduced PC number in co-cultures; these effects were reversed after transfecting EC with small interfering RNA targeted to PLA₂. PLA₂ and COX-2 protein expressions were significantly increased in microvessels from retina of diabetic rats. Diabetic rats had also high retinal levels of VEGF, ICAM-1 and TNFα that were reduced by treatment with a cPLA₂ inhibitor. In conclusion, the present findings indicate that PLA₂ upregulation represents an early step in glucose-induced alteration of BRB, possibly upstream of VEGF; thus, PLA₂ may be an interesting target in managing diabetic retinopathy.     

A phospholipase A2 isolated from Lachesis muta snake venom increases the survival of retinal ganglion cells in vitro

We have previously showed that a phospholipase A₂ isolated from Lachesis muta snake venom and named LM-PLA₂-I displayed particular biological activities, as hemolysis, inhibition on platelet aggregation, edema induction and myotoxicity. In the present work, we evaluated the effect of LM-PLA₂-I on the survival of axotomized rat retinal ganglion cells kept in vitro, as well as its mechanism of action. Our results clearly showed that treatment with LM-PLA₂-I increased the survival of ganglion cells (100% when compared to control cultures) and the treatment of LM-PLA₂-I with p-bromophenacyl bromide abolished this effect. This result indicates that the effect of LM-PLA₂-I on ganglion cell survival is entirely dependent on its enzymatic activity and the generation of lysophosphatidylcholine (LPC) may be a prerequisite to the observed survival. In fact, commercial LPC mimicked the effect of LM-PLA₂-I upon ganglion cell survival. To investigate the mechanism of action of LM-PLA₂-I, cultures were treated with chelerythrine chloride, BAPTA-AM, rottlerin and also with an inhibitor of c-junc kinase (JNKi). Our results showed that rottlerin and JNK inhibitor abolished the LM-PLA₂-I on ganglion cell survival. Taken together, our results showed that LM-PLA₂-I and its enzymatic product, LPC promoted survival of retinal ganglion cells through the protein kinase C pathway and strongly suggest a possible role of the PLA₂ enzyme and LPC in controlling the survival of axotomized neuronal cells.     

Investigating the contribution of CYP2J2 to ritonavir metabolism in vitro and in vivo

Ritonavir, an HIV protease inhibitor, is successfully used for the prevention and treatment of HIV infections. Ritonavir pharmacokinetics are complicated by inhibition, induction and pharmacogenetics of cytochrome P450 (CYP) enzymes mediating its clearance. This investigation revealed that CYP2J2, along with CYP3A4/5 and CYP2D6, efficiently metabolizes ritonavir, and to a CYP2J2-specific (minor) metabolite. Chemical inhibition of ritonavir metabolism, clearance, K_I/k_inact and abundance of CYP2J2 in liver microsomes were evaluated and then applied to an in vitro–in vivo static scaling model to estimate the contribution of each isozyme, as a function of CYP abundance, activity, and genotype. Disposition of the CYP2J2-specific metabolite was also evaluated in vivo. In plasma, metabolite abundance was well above previously reported levels with circulating concentrations measured at 2 μM for the main hydroxylisopropyl metabolite. Ritonavir and metabolite plasma profiles were simulated using Simcyp^®. A modest (2–6%) contribution of CYP2J2 to ritonavir clearance is predicted which increases to more than 20% in subjects carrying CYP2D6 poor metabolizer polymorphisms and CYP3A4 irreversible inhibition. These results indicate that minor drug metabolizing enzymes could become quantitatively important in RTV clearance if main metabolic pathways are impeded.     

Dopamine uptake in striatal synaptosomes exposed to peroxidation “in vitro”

The uptake of [³H]dopamine by synaptosomes from rat brain striatum was biphasic, the rapid phase requiring less than 1 min for completion and the slower phase occuring over a few minutes. This uptake of labeled dopamine was enhanced by prior exposure of the synaptosomes to Fe²⁺, which gives rise to peroxidation of the synaptosomal lipids. Both lipid peroxidation and enhancement of dopamine uptake were unaffected by the presence of inhibitors of monoamine oxidase but were blocked by inclusion of o-phenanthroline in the preincubation medium. It is concluded that lipid peroxidation may be involved in certain neuropsychiatric disorders such as seizures evoked by exposure to high oxygen pressures.     

Interactions of aflatoxin B1 and blood components of various species in vitro: Interconversion of aflatoxin B1 and aflatoxicol in the blood

The fate of aflatoxin B₁ (AFB₁) in the blood of various species of animals was studied in vitro. Examination of the distribution of radioactivity in blood incubated with [¹⁴C]AFB₁ at 37°C showed that high levels of radioactivity were associated with blood cells. The radioactivity was readily removed from the blood cells by washing with fresh plasma, indicating loose binding of AFB₁ to blood cells. Most of the radioactivity in plasma was bound to protein. These results suggest that a large part of the AFB₁ in blood in vivo may be carried not only by the plasma proteins but also by the blood cells. When chloroform extracts of plasma of [¹⁴C]AFB₁-treated mouse, rat, duckling, and hamster blood were developed by thin-layer chromatography, high levels of radioactivity were found in both the AFB₁ region and the aflatoxicol (AFL) region. Incubation of blood with nonradioactive AFB₁ and AFL showed marked interconversion of AFB₁ and AFL in the blood of rats, hamsters, mice, and Mongolian gerbils, but not in the blood of guinea pigs, rhesus monkeys, squirrel monkeys, or humans. Interconversion occurred in red blood cell suspensions but not in plasma, indicating that the red blood cells are responsible for AFB₁-AFL interconversion in the blood.     

Viability of Saccharomyces cerevisiae cells following exposure to H2O2 and protective effect of minocycline depend on the presence of VDAC

Proteins involved in apoptosis are still a matter of debate. Therefore, we decided to check the effect of the presence of VDAC (voltage dependent anion selective channel) on viability of Saccharomyces cerevisiae cells following their exposure to H₂O₂ that is known to induce apoptosis both in S. cerevisiae and in mammalian cells. Mitochondria of S. cerevisiae contain only one channel-forming VDAC isoform (VDAC1), which simplifies studies on the channel. Using S. cerevisiae mutant depleted of VDAC1 (termed here VDAC) and the isogenic wild type, we have shown that VDAC is important for protection of S. cerevisiae cells against H₂O₂ treatment, particularly in exponential growth phase that is known to be more affected by H₂O₂. The increased viability of H₂O₂ pretreated exponentially growing cells containing VDAC was accompanied by clear changes of the cytosol redox state and was potentiated by minocycline, an antibiotic of the tetracycline family that displays cytoprotective potency. The protective effect of minocycline also coincided with distinct changes of cytosol redox state. Thus, we conclude that the ability to change the cytosol redox state following exposure to H₂O₂ or/and minocycline appears to be an intrinsic feature of exponentially growing cells (young cells) containing VDAC. Moreover, the ability seems to be crucial for both cell viability and protective effect of minocycline.     

Understanding the in vitro neuromuscular activity of snake venom Lys49 phospholipase A2 homologues

Phospholipases A₂ (PLA₂s) with a lysine substituting for the highly conserved aspartate 49, Lys49 PLA₂ homologues, are important myotoxic components in venoms from snakes of Viperidae family. These proteins induce conspicuous myonecrosis by a catalytically-independent mechanism. Traditionally, the Lys49 PLA₂ homologues are classified as non-neurotoxic myotoxins given their inability to cause lethality or paralytic effects when injected in vivo, even at relatively high doses. However, a series of in vitro studies has shown that several Lys49 PLA₂ homologues from Bothrops snake venoms induce neuromuscular blocking activity on nerve-muscle preparations in vitro. The interpretation of these findings has created some confusion in the literature, raising the question whether the Lys49 PLA₂ homologues present some neurotoxic activity. The present article reviews the in vitro neuromuscular effects of Lys49 PLA₂ homologues and discusses their possible mechanisms of action. It was concluded that the neuromuscular blockade induced by Lys49 PLA₂ homologues in isolated preparations is mainly a consequence of the general membrane-destabilizing effect of these toxins.     

红车轴草总黄酮对H_2O_2诱导的血管内皮细胞损伤的保护作用

目的观察红车轴草总黄酮对H2O2诱导的血管内皮细胞损伤的保护作用,并探讨其可能的作用机制。方法利用体外培养的人脐静脉血管内皮细胞ECV-304,采用H2O2诱导细胞损伤模型,观察红车轴草总黄酮对H2O2所致的血管内皮细胞损伤的保护作用。MTT法检测细胞存活率、试剂盒测定细胞中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)活性及细胞中丙二醛(MDA)的含量、乳酸脱氢酶(LDH)的漏出量;荧光法检测Caspase-3的活性;Hoechst33342及PI荧光染色法观察红车轴草总黄酮对H2O2诱导血管内皮细胞凋亡的影响;JC-1染色法检测红车轴草总黄酮对H2O2损伤血管内皮细胞线粒体膜电位的影响,检测各组细胞内ROS的表达水平。结果与空白对照组相比,H2O2损伤组细胞存活率降低、细胞内LDH,漏出量增加(P<0.001);抗氧化酶SOD、CAT、GSH-Px活性降低、MDA含量升高(P<0.01);Caspase-3活性升高(P<0.001)、ROS含量升高、线粒体膜电位降低;与模型组相比,红车轴草总黄酮各剂量组(12.5、25、50mg.L-1)及槲皮素组(30 mg.L-1)细胞存活率明显增加(P<0.01,P<0.05),红车轴草总黄酮各剂量组(12.5、25、50 mg.L-1)及槲皮素组(30 mg.L-1)均能明显降低LDH漏出量(P<0.01,P<0.05)、明显增强SOD、CAT及GSH-Px活力(P<0.01,P<0.05)、降低MDA、ROS含量及Caspase-3活性(P<0.05,P<0.01)、升高线粒体膜电位(P<0.01)。结论红车轴草总黄酮对H2O2诱导的内皮细胞损伤有明显的保护作用,其初步作用机制可能与抗自由基,减轻脂质过氧化、降低Caspase-3活性及稳定线粒体膜电位有关。     

LL-202, a newly synthesized flavonoid,inhibits tumor growth via inducing G2/M phase arrest and cell apoptosis in MCF-7 human breast cancer cells in vitro and in vivo

We recently established that LL-202, a newly synthesized flavonoid, exhibited obvious anticancer effects against human breast cells in vivo and in vitro. The underlying mechanism of its anticancer activity remains to be elucidated. In this study, we demonstrated that LL-202 inhibited the growth and proliferation of human breast cancer MCF-7 cells in a concentration and time-dependent manner. We reported that LL-202 induced both mitochondrial- and death-receptor-mediated apoptosis, which were characterized by the dissipation of mitochondrial membrane potential (ΔΨ_m), cytochrome c (Cyt c) release from mitochondria to cytosol, the activation of several caspases and induction of poly (ADP-ribose) polymerase (PARP) and Bid cleavage. N-acetylcysteine (NAC), a general ROS scavenger, partly blocked the LL-202-induced ROS levels and apoptosis. In addition, LL-202 induced arrest in cell cycle progression at G2/M phase in MCF-7 cells. After the treatment with LL-202, the expression of cell cycle-related proteins, such as cyclin B1, cyclin A, and p-CDK1 (Thr161) were down-regulated, whereas the expression of p21^WAF1/Cip1 and p-CDK1 (Thr14/Tyr15) were up-regulated. Finally, in vivo studies, LL-202 significantly suppressed the growth of MCF-7 breast cancer xenograft tumors in a dose-dependent manner with low systemic toxicity. In conclusion, the results showed that LL-202 had significant anticancer effects against human breast cells via the induction of apoptosis and G2/M phase arrest and it may be a novel anticancer agent for treatment of breast cancer.     

The toxicity of H2O2 on the ionic homeostasis of airway epithelial cells in vitro

Oxygen species may be formed in the air spaces of the respiratory tract in response to environmental pollution such as particulate matter. The mechanisms and target molecules of these oxidants are still mainly unknown but may involve modifications of the ionic homeostasis in epithelial cells. Cytosolic concentrations of Ca²⁺ (Fura2) and Na⁺ (SBFI) and short-circuit current (Isc) were followed in primary cultures of human nasal epithelial cells and in the cell line 16HBE14o⁻ after exposure to H₂O₂ or ·OH (H₂O₂+Fe²⁺). Cells were grown on glass coverslips for ionic imaging or on permeable snapwell inserts for Isc studies. Exposure of the apical as well as the basal side of the cultures to H₂O₂ or ·OH induced a concentration-dependent transient increase in Isc which is due to a transient secretion of Cl⁻. Ca_i also increased transiently with approximately the same kinetics. The response was dependent on the release of calcium from intracellular stores. Na_i on the contrary increased steadily over more than an hour. When the apical membrane was permeabilized with gramicidin, ·OH inhibited the Na⁺ current (a measure of Na⁺-K⁺-ATPase activity in the baso-lateral membrane). The arrest of the pump was significant after 30 min exposure to oxidant. On the other hand no increase in the apical or baso-lateral sodium conductances could be detected. The progressive arrest of the Na⁺/K⁺-pump may contribute to the sustained elevation of Na_i. This strong modification in the cellular ionic homeostasis may participate in the stress response of the respiratory epithelium through alterations in signal transduction pathways.     

人参蛋白协同H2O2诱导SH-SY5Y细胞氧化损伤

目的:探讨人参蛋白(GP)协同H₂O₂诱导人神经母细胞瘤(SH-SY5Y)氧化应激损伤的作用,并筛选二者的最佳联合浓度。方法:首先采用不同浓度H₂O₂诱导SH-SY5Y细胞氧化损伤,然后采用不同浓度GP联合200 μmol·L^-1H₂O₂诱导SH-SY5Y细胞氧化损伤;采用倒置荧光显微镜观察细胞形态,MTT法检测细胞存活,Hoechst 33342染色法检测细胞凋亡。结果:SH-SY5Y细胞经GP-H₂O₂作用后,细胞数量减少,轴突缩短或消失,胞体变圆、缩小,大小不等;细胞存活率降低;Heochst 33342染色呈现高蓝光;GP 60 mg·L^-1+H₂O₂ 200 μmol·L^-1为联合诱导氧化应激损伤的最佳浓度。结论:GP有协同H₂O₂诱导SH-SY5Y细胞氧化损伤的作用,抑制细胞增殖、促进细胞凋亡是其可能的作用机制。     

Role of hMLH1 in sterigmatocystin-induced G2 phase arrest in human esophageal epithelial Het-1A cells in vitro

Sterigmatocystin (ST), a common environmental contaminant found across the world, is generally recognized as a potential carcinogen, mutagen and teratogen. Our previous epidemiological studies suggested that ST exposure might be a risk factor for esophageal cancer. However, the direct effects of ST on human esophageal epithelial cells are currently unknown. In the present study, we examined the effect of treating a human esophageal epithelial cell line (Het-1A) with ST on DNA damage, DNA repair mechanisms, and cell cycle distribution. We found that ST treatment could induce DNA damage and lead to a G₂ phase arrest, associated with a marked up-regulation of G₂/M regulatory proteins, including Cyclin B1, Cdc2/p-Cdc2, and Cdc25C/p-Cdc25C. Additionally, we found that the expression of two mismatch repair (MMR) proteins, hMLH1 and hMSH2, was up-regulated at both the mRNA and protein levels after ST treatment, suggesting that ST could induce the MMR system in Het-1A cells. Interestingly, ST-induced G₂ phase arrest was mediated by hMLH1 up-regulation, but was independent of hMSH2. Treatment with hMLH1-siRNA prevented the up-regulation of Cyclin B1, Cdc2/p-Cdc2 and Cdc25C/p-Cdc25C in ST-treated cells, thereby inhibiting the subsequent G₂ phase arrest of Het-1A cells. Moreover, we found that hMLH1 may act as a direct sensor of ST-mediated DNA damage. In conclusion, the study demonstrated that ST caused DNA damage and triggered G₂ phase arrest in Het-1A cells, and hMLH1 participated in the ST-induced G₂ phase arrest by up-regulating G₂/M regulatory proteins.     

曲美他嗪对内皮祖细胞氧化应激损伤的保护作用研究

目的: 研究曲美他嗪(TMZ)对过氧化氢(H₂O₂)诱导的内皮祖细胞(EPCs)氧化应激损伤的保护作用。方法: 采用密度梯度离心法分离人脐带血单个核细胞,使用EBM-2完全培养基诱导单个核细胞向内皮祖细胞分化,经细胞形态学变化及流式细胞术测定内皮祖细胞特异性标记物CD133、CD34和VEGFR抗原来鉴定内皮祖细胞。体外建立内皮祖细胞过氧化氢(100 μmol·L^-1)损伤模型,加入不同浓度曲美他嗪(0.1,0.5,1,5 μmol·L^-1),观察曲美他嗪作用不同时间后(0.5,1,6,12 h)内皮祖细胞增殖、黏附、迁移、一氧化氮(NO)分泌能力的变化。结果: 曲美他嗪呈剂量及时间依赖性降低过氧化氢对内皮祖细胞的氧化应激损伤,保护内皮祖细胞增殖、黏附、迁移、分泌的生物学功能。结论: 曲美他嗪在氧化应激条件下对内皮祖细胞生物学功能的保护可能是其心血管保护作用的机制之一。     

Activation of P53 in HepG2 cells as surrogate to detect mutagens and promutagens in vitro

The current genotoxicity tests of the standard in vitro battery, especially those using mammalian cells, are limited by their low specificity and highlight the importance of new in vitro tools. This study aimed to evaluate the suitability of HepG2 cells for assaying mutagens and promutagens. We determined P53 activity as surrogate genotoxicity endpoint in HepG2 cells. Our results revealed a significant P53-induction by actinomycin D, methyl methanesulfonate and etoposide. Prior to the investigation of promutagens we characterized HepG2 cells by analyzing the expression of 45 genes involved in xenobiotic metabolism and measuring the activity of selected Cytochrome-P450 (CYP) enzymes. We determined a limited metabolic capacity prompting us to employ a co-treatment with rat liver S9 as metabolic activation system (MAS) for promutagens. While cyclophosphamide showed an elevation of activated P53 in the presence of S9, 7,12-dimethylbenz[a]anthracene and aflatoxin B₁ responded without the MAS. Inhibition of cellular CYP3A4 or CYP1A/1B suppressed the aflatoxin B₁- and dimethylbenz[a]anthracene-mediated P53 response, respectively, indicating that HepG2 cells are capable of metabolizing these compounds in a CYP1A/B/3A4-dependent manner. In summary, our results indicate that P53 activation in HepG2 cells combined with a MAS can be used for the identification of new (pro)genotoxicants.     

人参皂苷Rg3对H2O2诱导人肾小球系膜细胞氧化应激损伤的保护作用及其机制研究

目的探究人参皂苷Rg_3对H_2O_2诱导人肾小球系膜细胞氧化应激损伤的保护作用及其作用机制。方法采用H_2O_2诱导建立人肾小球系膜细胞氧化应激损伤模型。以不同浓度的人参皂苷Rg_3对人肾小球系膜细胞预处理24 h,采用CCK-8试验、乳酸脱氢酶(LDH)试剂盒以及丙二醛(MDA)测定试剂盒分别检测细胞凋亡数量、细胞培养液中LDH水平和MDA含量。通过流式细胞术、q RT-PCR以及Western blotting检测细胞周期、CDK4的m RNA水平和蛋白表达。结果 400μmol/L H_2O_2培养细胞4 h能够建立稳定有效的人肾小球系膜细胞氧化应激损伤模型。随着人参皂苷Rg_3浓度的增加,被H_2O_2损伤的细胞的凋亡数量、细胞外LDH活性、细胞产生的MDA的含量均明显下降。40μmol/L人参皂苷Rg_3可以把人肾小球系膜细胞的细胞周期阻滞在G1期,下调CDK4的m RNA水平和蛋白表达水平。结论人参皂苷Rg_3可以通过抑制CDK4而对人肾小球系膜细胞氧化应激损伤发挥保护作用。     

浒苔多糖对H_2O_2诱导的PC12细胞损伤的保护作用

目的研究浒苔多糖对H2O2诱导的PC12细胞损伤的保护作用。方法用水提法提取浒苔多糖,除蛋白后用DE-52离子交换柱进行纯化。用MTT法检测纯化的浒苔多糖对H2O2诱导的PC12细胞损伤的保护作用。结果纯化的浒苔多糖能明显改善H2O2导致的细胞损伤,与H2O2处理组相比,浒苔多糖100,200和300μg/mL组可使细胞存活率升高。结论浒苔多糖对H2O2诱导的PC12细胞损伤具有明显的保护作用。